1. Field of the Invention
The present invention relates to novel C60 derivatives, processes for preparing such derivatives and methods of treatment. In more detail, disclosed and claimed herein are (a) novel e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene and processes for preparing the same, (b) compositions and methods of treating neuronal injury with a therapeutically effective amount of e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene and (c) compositions and methods for prolonging the length or duration of an expected lifespan (referred to alternately as “longevity”) of metazoans or in metazoan cells with a therapeutically effective amount of e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene.
2. Related Art
Methods of enhancing the overall health and longevity of humans and their companion animals has been a very active area of research. Current thinking in the field suggests that calorie restriction may help to extend the lifespan of metazoans.
Given the conserved nature of cellular or developmental processes across metazoans, a number of model organisms have been employed to study longevity, including C. elegans and D. melanogaster. 
For example, the genetic analysis of C. elegans has revealed several genes involved in lifespan determination. Mutations in Daf-2 (the insulin receptor) and Clk-1 (“Clock 1”, a gene affecting many aspects of developmental and behavioral timing) have been shown to extend the lifespan of adults. However, Clk-1 mutants have a higher mortality rate in early life. At later stages of development, the Clk-1 mutants show an increase in longevity, perhaps by selecting for long-lived individuals in early life. The Clk-1 longevity phenotype is abolished by mutations in the gene encoding catalase, which is involved in superoxide/free radical metabolism. Additionally, elimination of coenzyme Q in C. elegans diet has been shown to extend lifespan.
C. elegans harboring mutations in the Eat gene have also shown an increased longevity, but exhibit decreased food intake and slowed metabolism. The enhanced longevity associated with this mutation has been attributed to calorie restriction, which has been shown to also increase longevity in metazoans.
In Drosophila, superoxide dismutase (SOD) and catalase over expression increased the lifespan of fruit flies by 35%. Mutations also in the Methuselah gene (“Mth”) have been shown to increase lifespan by 20%. The function of Mth, a G-protein coupled receptor, is not known, but mutants have shown an increased resistance to paraquat (a superoxide radical injury inducing agent) toxicity, suggesting it may be a stress-response gene.
Calorie restriction (CR) has been shown to increase lifespan by 25–35% in all animals studied to date (mice, rats, several species of monkeys, dogs, humans, as well as non-metazoan species such as spiders, Nematodes, and Drosophila). (NB: All animals are metazoans.) However, caloric intake needs to be reduced by as much as 30–40% to achieve robust effects on longevity. Ongoing studies in rhesus and squirrel monkeys at the National Institute of Aging (“NIA”) (Roth et al., Eur. J. Clin. Nutr. S:157, 2000) found biochemical changes in calorie restricted monkeys similar to changes reported in rodents thereby supporting the universal nature of calorie restriction on biochemical processes across vertebrate species.
Recently, 2-deoxyglucose has been used to produce calorie restriction without limiting oral intake. Animals treated with 2-deoxyglucose have lowered body temperature and decreased plasma insulin levels, similar to changes observed in calorie-restricted animals (Roth et al., Ann. NY Acad. Sci., 928: 305, 2001). While scientific studies on the effect of 2-deoxyglucose on longevity have not been completed, a recent editorial in Science (Feb. 8, 2002) quoted the principal investigator of these studies (George Roth, NIA) as saying that one of his monkeys treated with 2-deoxyglucose lived 38 months instead of the mean survival of 25 months. However, such a claim is not scientifically supported given the small sample size. No comment was made on the age of the longest-lived monkeys in the control populations.
Increases of up to 20% in the expected lifespan of mice has been shown through growth factor deprivation, either through genetic manipulation or the administration of growth factor antagonists. Unfortunately, dwarfism is a side effect of growth factor deprivation. In humans, dwarfism, or late-life growth hormone deficiency, appears to reduce longevity, further confusing the issue of whether growth factor deprivation is effective as a means for increasing the duration of the expected lifespan.
Several papers have indicated that deprenyl (a selective monoamine oxidase (MAO) B inhibitor used to treat Parkinson's disease) increases the lifespan of many species. (See, e.g. Knoll, Mech Ageing Dev. 46:237, 1988). In one study, chronic treatment of rats with deprenyl from age 96 weeks through the end of life “enhanced survival”. Control rats lived 147+/−1 weeks, whereas the deprenyl-treated rats lived 198+/−2 weeks. However, the expected mean lifespan for these rats, clearly stated in the paper, was 182 weeks, so the control group in this study appears to have had early mortality. Other studies from these laboratories selected for high-performing rats, which were then enrolled in the deprenyl longevity studies, thereby potentially artificially skewing the results.
A second study used Fisher 344 rats (Kitani et al., Life Sci 52:281, 1993), initiating deprenyl treatment at 18 months of age. The mean survival of the controls was 28 months, and of the treated animals was 30 months, showing an increase in longevity of 7%. However, these results were shown to be not statistically significant.
In contrast, another study in Fisher 344 rats with the same dose of deprenyl (Carillo et al, Life Sci 67:2539, 2000), observed greater mortality and shortened lifespan in the deprenyl-treated animals. Furthermore, a study from the NIA failed to show any survival benefit in C57B6 mice given chronic deprenyl treatment starting at 18 months of age (Ingram et al., Neurobiol Aging 14:431, 1993). Likewise, a controlled study of deprenyl in Drosophila did not show an increase in lifespan (Jordens et al., Neutrochem Res 24:227, 1999).
Human trials of deprenyl likewise show conflicting results regarding longevity. An “open, uncontrolled” trial of deprenyl in Parkinson's patients showed an increase survival at 9 years (Birkmayer et al., J Neural Transm. 64:113, 1985), although other studies have suggested increased mortality in PD patients taking deprenyl, especially in conjunction with L-dopa (e.g. Ben-Shlomo et al., BMJ 316:1191, 1998).
Overall, the data suggest that deprenyl may or may not have weak effects on longevity.
Several genes in mice have been identified as “longevity” genes because mice with mutations in these genes have greater mean lifespans relative to the expected lifespan of control mice. These genes include the Ames dwarf mutation, and the Snell dwarf mutation. However, these mutations result in small, frail mice which have difficulty feeding. It is believed that the longevity conferred by these mutations is essentially due to calorie restriction. Recent attempts to use gene array analysis, or other genetic screens for genes associated with longevity phenotypes in worms, flies, and rodents have come up with a number of candidate genes. In general, however, they are frequently “stress-response” genes.
Many compounds, such as Gingko, Ginseng, Vitamin C, have been proposed to improve survival, but controlled and statistically significant survival studies reporting the benefit for these compounds are unknown. Vitamin C and a number of drugs reduce the incidence of certain disease conditions, e.g. cardiovascular disease, and so, presumably, would enhance overall longevity.
Buckminsterfullerene, C60, is a carbon sphere with 12 pentagons and 20 hexagons, soluble in aromatic solvents but not in water.
Use of C60(C(COOH)2)n, wherein n is an integer from 1 to 4, is disclosed for treating neuronal injury in U.S. Pat. No. 6,265,443, issued Jul. 24, 2001 to Choi et al., incorporated herein by reference in its entirety.
The preparation of C3 hexacarboxylic (“C3” or “Hexa”) acid reported in the literature produces mixtures of products, some unidentified, with poor reproducibility and variable performance on cell culture screening.